The human oral cavity is inhabited by hundreds of bacterial species, most of which are commensal species, necessary to maintain equilibrium in the oral ecosystem. However, some of them play a key role in the development of oral diseases, primarily dental caries and periodontal disease (1). Oral diseases begin with the growth of dental plaque, a biofilm formed by the accumulation of bacteria jointly with glycoproteins from human saliva and polysaccharides secreted by microbes (2). The subgingival plaque, located in the neutral or alkaline subgingival pocket, is typically inhabited by Gram-negative anaerobes and is responsible for the development of gingivitis and periodontitis. The supragingival dental plaque is formed on the surface of the tooth and includes acidogenic and acidophilic bacteria, which, upon fermenting the sugars ingested in the diet, produce acid and lower the pH. When the pH is too acidic (generally with a value less than 5.5), the tooth enamel is de-mineralised and destroyed, and, therefore, these bacteria are responsible for dental caries, which are considered to be the most widespread infectious disease in the world, affecting over 80% of the human population (3). A bad oral health may be associated with other pathologies, such as, for example, stomach ulcers, stomach cancer or cardiovascular diseases, amongst others.
One of the main reasons why, as of today, oral pathogens have still not been eradicated is the difficulty involved in studying microbial communities that inhabit the oral cavity, since, on the one hand, the complexity of the ecosystem (several hundreds of species have been detected, with numerous levels of interaction) makes it difficult to detect the potential pathogenic species (4); moreover, no single etiological agent may be identified, as in classic diseases, following Koch's postulates. This fact has been clearly demonstrated in periodontal disease, where there are at least 3 bacterial species belonging to very different taxonomic groups (the so-called “red complex” of periodontal pathogens) which have been associated with the development and progression of periodontal disease (5). On the other hand, a large proportion of oral bacteria cannot be cultured (6) and, therefore, traditional microbiological methods give an incomplete image of the natural communities that inhabit dental plaque. However, the current development of metagenomic techniques and Next-Generation Sequencing technologies allows for the study of the bacterial community as a whole, by analysing the total DNA of complex microbial samples (Metagenome) without the need to culture the bacteria themselves.
In this regard, pioneering studies in metagenomics have focused on the intestinal ecosystem primarily through a shot-gun approach, wherein the DNA is cloned in small-size plasmids, followed by traditional Sanger-type sequencing (7, 8). More recent approaches include sequencing of the ends of large-size fosmids (9) and use of the “Illumina” sequencing technology, which provides a high coverage of short sequences (10). Studies of the microbiota of the oral cavity, as well as of other human body habitats, such as the skin, the vagina or the respiratory tract, have focused on the sequencing of ribosomal RNA amplicons (11, 12). These studies have provided a substantial improvement of our knowledge about these bacterial communities as compared to prior research based on cultures, but estimates of microbial diversity are hindered by the biases in PCR amplification (i.e. PCR only detects the bacteria that are most similar to those already known, and on the basis of which the amplification primers are used, giving an incomplete image of the diversity present), the cloning bias (a large number of genes are not cloned because they are toxic for the host bacteria and, therefore, this method does not allow for the study of the entire genetic reservoir of the sample) and a low sequence length (the sequences in the Illumina technology have only between 35-70 nucleotides, which in most cases makes a reliable taxonomic or functional assignment impossible), together with the fact that, as mentioned above, a large proportion of oral bacteria cannot be cultured.
In order to resolve the aforementioned problems, the present invention discloses the obtainment of the metagenome of dental plaque by the direct sequencing of metagenomic DNA, using 454-pyrosequencing, thereby eliminating the potential biases imposed by cloning and PCR techniques, and, furthermore, providing access to the entire genetic repertoire of the oral bacterial community under different health conditions, as well as the possibility to analyse which bacterial species amongst those found in the metagenome obtained may be associated with a good oral health, since those individuals who had never suffered from caries exhibit a different bacterial flora than those individuals who had suffered or currently suffer from it. By means of the oral metagenome obtained in the present invention, it is possible to direct the isolation, culture and identification of strains with anti-cariogenic activity from the conglomerate of bacteria in the buccal cavity sample; specifically, the supragingival plaque of individuals who have never suffered from caries, i.e. those strains capable of inhibiting the growth of cariogenic bacteria.
Another strategy disclosed in the present invention is the obtainment of a metagenomic library of fosmids (long DNA inserts, approximately 35-45 Kb) from the dental plaque of individuals who have never suffered from caries. By obtaining said fosmid library, it is possible to isolate and identify the bioactive anti-cariogenic peptides synthesised by the bacteria present in the oral cavity of individuals who have never suffered from caries. In this regard, given that, in the state of the art, Streptococcus mutans has been shown to be the main causal agent of caries (13), it is not surprising that most strategies against this disease have been aimed against said microorganism. These strategies have included the development of vaccines using known surface antigens, passive immunisation strategies that may neutralise the bacteria, the co-aggregation of S. mutans with probiotic strains and the use of inhibitory proteins specific to S. mutans, amongst others (14).
Other different strategies have been those disclosed in different patent documents, which propose the use of different bacterial strains, preferably S. mutans, that produce a lower concentration of acid (15), or the use of the same resources, for example, the nutrients, by pathogenic strains and non-pathogenic strains, continuously supplying high concentrations of non-pathogenic bacteria, which results in the displacement of the pathogenic bacteria, provided that they share the same resource (16), or even a lower adherence of cariogenic bacterial strains to the tooth (17). On the contrary, the bioactive strains and peptides disclosed in the present invention have antibiotic activity, preferably anti-cariogenic activity, on their own, against caries-producing microorganisms. On the other hand, patent WO20040072093 (18) discloses a number of anti-microbial agents that are active primarily against Gram-negative microorganisms, but the main causal agents of caries, S. mutans and S. sobrinus, are Gram-positive microorganisms. Moreover, the S. mitis and S. oralis isolates that produce the anti-microbial peptides disclosed in WO20040072093 (18) have been isolated from the throat of patients with cystic fibrosis, not from the mouth of people without caries, as in the case of the peptides and/or strains of the invention. Similarly, the therapeutic use of said peptides is aimed at the treatment of respiratory tract diseases and not of caries, as in the case of the bioactive peptides disclosed in the present invention.
In this regard, the main technical characteristics that make the bacterial strains isolated and disclosed in the present invention different from the rest of strains disclosed in the state of the art are that they may be cultured by means of conventional microbiological techniques; that they present inhibitory activity against organisms that produce infectious diseases of the buccal cavity, preferably caries, without the need to be genetically modified; and that they have been isolated from individuals who have never suffered from caries. Consequently, both the anti-cariogenic bacteria themselves and the bioactive anti-cariogenic compounds, preferably peptides, disclosed in the present invention may be used as probiotic and/or prebiotic compositions as such, or as a part of different pharmaceutical compositions used for the treatment of infections of the buccal cavity, such as, for example, caries, periodontitis, etc., or even as functional foods. Moreover, the present invention also discloses a method for the prevention and/or treatment of infectious diseases of the buccal cavity, preferably caries, that comprises the administration of a pharmaceutically effective quantity of at least one of the strains and/or at least one of the anti-microbial compounds, preferably peptides, described above, or of the probiotic or pharmaceutical composition or functional foods that comprise at least one of the strains and/or at least one of the compounds, preferably peptides, of the invention.